Direct cytosolic communication between cells is mediated by an aggregate of intercellular channels in the plasma membrane called the gap junction (GJ). Remodeling of connexin43 (Cx43) GJs has been linked to cardiac arrhythmias. Work accomplished during the previous period of this RO1 identified a biological function of interaction between Cx43 and the actin-binding protein Zonula Occludens (ZO)-1 in GJ remodeling. In recent work, we have determined that a peptide known to inhibit Cx43/ZO-1 interaction, 1CT1, reduces the frequency of inducible arrhythmias following injury to the left ventricle. Preliminary data from work performed with 1CT1 in vitro, indicates that the transition from free connexon channels in the membrane to paired connexons in intercellular channel aggregates underlies GJ remodeling. We hypothesize that ZO-1 regulates the rate at which free connexons in the membrane accrete to GJs-the "connexon switch". The aims in this renewal will test this hypothesis and its implications for GJ intercellular communication, membrane excitability, and a novel role for Cx43 in differential adhesion between myocytes and fibroblasts following myocardial infarction - all processes likely to impact susceptibility to re-entrant arrhythmia. The proposed experiments will quantitatively determine the role of Cx43/ZO-1 interaction in the "connexon switch" using methods including live cell imaging, fluorescent fusion proteins, loss-of-function mutants, whole cell patch clamp, single channel electrophysiology and biochemical assays. Implications of Cx43/ZO-1 interaction within the physiological framework of cardiac injury in vivo will be assessed by echocardiography, EKG telemetry, optical mapping of electrical activation and arrhythmia induction protocols. The data generated will broaden our understanding of fundamental mechanisms of Cx43 function and may translate to new therapies for the prevention and treatment of cardiac arrhythmia. PUBLIC HEALTH RELEVANCE: Injury and scarring of the heart following myocardial infarction (heart attack) is one the most frequent causes of sudden death in the USA. This project will deliver new insights on the function of a protein essential to a stable heartbeat called connexin43. The project aims to provide novel approaches to the prevention and treatment of heart attacks.